Patent application title: MODIFIED ACTIVIN A
Inventors:
IPC8 Class: AC07K1452FI
USPC Class:
1 1
Class name:
Publication date: 2021-09-02
Patent application number: 20210269495
Abstract:
The purpose of the present invention is to provide a modified activin A.
The present invention provides activin A comprising a modified proregion.Claims:
1. Activin A having resistance to degradation by a protease other than
Furin.
2. The activin A according to claim 1, wherein the protease is an endogenous plant protease.
3. The activin A according to claim 1, wherein the activin A comprises a modified proregion.
4. The activin A according to claim 3, wherein the modified proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and/or added in an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6.
5. Activin A, comprising a modified proregion, wherein the modified proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and/or added in an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6.
6. The activin A according to claim 5, wherein the modified proregion comprises any amino acid sequence selected from the group consisting of (a) to (d): (a) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is deleted; (b) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a spacer sequence consisting of 1 to 10 amino acids; (c) an amino acid sequence in which at least one of amino acids from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a different amino acid; and (d) an amino acid sequence in which an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 has at least one amino acid inserted therein.
7. The activin A according to claim 6, wherein the amino acid constituting the spacer sequence in the amino acid sequence (b) is at least one amino acid selected from the group consisting of glycine, alanine, and serine.
8. The activin A according to claim 6, wherein the different amino acid in the amino acid sequence (c) is selected from the group consisting of alanine, serine, glycine, valine, leucine, and isoleucine.
9. The activin A according to claim 5, wherein the activin A has resistance to degradation by a protease other than Furin.
10. The activin A according to claim 5, wherein the modified proregion comprises any of amino acid sequences selected from the group consisting of (e) to (g): (e) an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20; (f) an amino acid sequence in which one or several amino acids are deleted, substituted or added in an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence has resistance to degradation by Furin; and (g) an amino acid sequence having 80% or more homology with an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence has resistance to degradation by Furin.
11. A polynucleotide encoding the activin A according to claim 1.
12. A vector comprising the polynucleotide according to claim 11.
13. A transformant expressing the activin A according to claim 1.
14. The transformant according to claim 13, wherein the transformant is a plant transformant.
15. The transformant according to claim 14, wherein the transformant expresses the activin A in apoplast.
16. A method for producing activin A, the method comprising: recovering the activin A expressed in the transformant according to claim 13.
17. A method for producing activin A, the method comprising: (a) recovering the activin A expressed in the transformant according to claim 13; and (b) treating the obtained activin A with Furin.
18. Activin A that is a mixture of (i) activin A comprising a modified proregion and (ii) mature activin A, wherein the modified proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and/or added in an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6.
Description:
TECHNICAL FIELD
[0001] The present invention relates to activin A comprising a modified amino acid sequence.
BACKGROUND ART
[0002] Activin A is a cytokine belonging to the TGF.beta. (transforming growth factor-.beta.) superfamily. Activin A is a physiologically and industrially useful protein that is involved in development and differentiation as an erythroid differentiation factor and a mesoderm-inducing factor and regulates diverse functions in various cells.
[0003] Examples of known methods for producing protein include a production method by transiently expressing a protein of interest in a plant (Non Patent Literature 1: Sainsbury et al., Curr. Opin. Plant Biol., 19, 1-7, 2014) and a method of expressing a target protein in apoplast (Patent Literature 1: JP Laid-Open Publication No. 2005-501558).
[0004] However, it has been reported that when a target protein is expressed in a plant, the expressed target protein is degraded by an endogenous plant protease, causing the problem of a reduced yield (Non Patent Literature 2: Mandal MK. et al., Front Plant Sci., 7, 267, 2015). In particular, there are abundant proteases having various spectra (ranges) of specificities in apoplast (Non Patent Literature 3: Pillary P. et al., Bioengineered, 5: 1, 15-20, 2014). Furthermore, the site of the target protein recognized by a protease varies depending on the type of protein, and thus, it is difficult to predict the site. Known methods to address these problems include a method of changing protein localization by using a protein sorting signal and a method of coexpressing a protease inhibitor in a plant (Non Patent Literature 2 and Non Patent Literature 3).
[0005] Some conformations of activin A have been reported (Non Patent Literature 4: Wang X. et al., Nat. Commun. 2016 Jul. 4; 7: 12052). The structural information about the site recognized by a protease other than a proprotein convertase (Furin) is unknown.
CITATION LIST
Patent Literature
[0006] [Patent Literature 1]
[0007] JP Laid-Open Publication No. 2005-501558
Non Patent Literature
[0007]
[0008] [Non Patent Literature 1]
[0009] Sainsbury et al., Curr. Opin. Plant Biol., 19, 1-7, 2014
[0010] [Non Patent Literature 2]
[0011] Mandal MK. et al., Front Plant Sci., 7, 267, 2015
[0012] [Non Patent Literature 3]
[0013] Pillary P. et al., Bioengineered, 5: 1, 15-20, 2014
[0014] [Non Patent Literature 4]
[0015] Wang X. et al., Nat. Commun. 2016 Jul. 4; 7: 12052
[0016] [Non Patent Literature 5]
[0017] Koretz K. et al., Histochemistry, 86: 5, 471-8, 1987
SUMMARY OF INVENTION
Technical Problem
[0018] As for activin A, conventionally known methods have not yet solved the problem of degradation by a protease other than a proprotein convertase (Furin) (hereinafter, may be simply referred to as "protease"). Accordingly, a novel activin A that is hard to be degraded by the above described protease (has resistance to degradation by the above described protease) is needed.
Solution to Problem
[0019] The present inventors conducted dedicated research to solve the above described problems and have found an unknown recognition site for proteases on activin A. Furthermore, the present inventors modified the amino acid sequence of a proregion of activin A, thereby successfully generating a modified activin A that is hard to be degraded by the protease, and thus, have accomplished the present invention.
[0020] Specifically, embodiments of the present invention are as follows:
(1) Activin A having resistance to degradation by a protease other than a proprotein convertase (Furin). (2) The activin A according to the above described (1), wherein the protease other than a proprotein convertase (Furin) is an endogenous plant protease. (3) The activin A according to the above described (1) or (2), wherein the activin A comprises a modified proregion. (4) The activin A according to the above described (3), wherein the amino acid sequence of the modified proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and/or added in an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6. (5) Activin A comprising a modified proregion, wherein the amino acid sequence of the modified proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and/or added in the amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6. (6) The activin A according to the above described (5), wherein the amino acid sequence of the modified proregion comprises any amino acid sequence selected from the following (a) to (d):
[0021] (a) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is deleted;
[0022] (b) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a spacer sequence consisting of 1 to 10 amino acids;
[0023] (c) an amino acid sequence in which at least one of amino acids from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a different amino acid; and
[0024] (d) an amino acid sequence in which an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 has at least one amino acid inserted therein. (7) The activin A according to the above described (6), wherein the amino acid constituting the spacer sequence in the above (b) is at least one amino acid selected from glycine, alanine, and serine. (8) The activin A according to the above described (6), wherein the different amino acid in the above (c) is one selected from alanine, serine, glycine, valine, leucine, and isoleucine. (9) The activin A according to any of the above described (5) to (8), wherein the activin A has resistance to degradation by a protease other than a proprotein convertase (Furin). Herein, the protease other than a proprotein convertase (Furin) is, for example, an endogenous plant protease. (10) The activin A according to the above described (5), wherein the amino acid sequence of the modified proregion comprises any of amino acid sequences selected from the following (e) to (g):
[0025] (e) an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20;
[0026] (f) an amino acid sequence in which one or several amino acids are deleted, substituted or added in an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence has resistance to degradation by a protease other than a proprotein convertase (Furin); and
[0027] (g) an amino acid sequence having 80% or more homology with an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence has resistance to degradation by a protease other than a proprotein convertase (Furin). (11) A polynucleotide encoding the activin A according to any of the above described (1) to (10). (12) A vector comprising the polynucleotide according to the above described (11). (13) A transformant expressing the activin A according to any of the above described (1) to (10). (14) The transformant according to the above described (13), wherein the transformant is a plant. (15) The transformant according to the above described (14), wherein the transformant expresses the activin A in apoplast. (16) A method for producing activin A, comprising the step of recovering the activin A expressed in the transformant according to any of the above described (13) to (15). (17) A method for producing activin A, comprising the following steps:
[0028] (a) recovering the activin A expressed in the transformant according to any of the above described (13) to (15); and
[0029] (b) treating the obtained activin A with a proprotein convertase.
Effects of Invention
[0030] The modified activin A of the present invention allows for improving the yield of activin A in producing the activin A in the presence of the protease other than a proprotein convertase (Furin) (for example, in planta), compared to a wild type activin A that is not modified.
BRIEF DESCRIPTION OF DRAWINGS
[0031] FIG. 1 is a schematic figure showing the structure of a human activin A.
[0032] FIG. 2 is an image demonstrating that degradation of a modified activin A by proteases was suppressed.
DESCRIPTION OF EMBODIMENTS
[0033] Hereinbelow, the present invention will be described in detail. The following embodiment is illustrated to describe the present invention and is not intended to limit the present invention to this embodiment. The present invention may be carried out in various modes without departing from the spirits of the present invention. This specification also encompasses the contents described in the specification and drawings of the Japanese patent application (Japanese Patent Application No. 2018-214630) filed on Nov. 15, 2018, to which the present application claims priority.
1. Summary
[0034] Activin A is a cytokine belonging to the TGF.beta. superfamily. Activin A is a physiologically and industrially useful protein that is involved in development and differentiation as an erythroid differentiation factor and a mesoderm-inducing factor and regulates diverse functions in various cells.
[0035] An exemplary method for producing a target protein is a method of producing the protein by transiently expressing the same in a plant. However, it has been reported that when a target protein is expressed in a plant, the expressed target protein is degraded by a protease other than a proprotein convertase (Furin) (for example, an endogenous plant protease), causing the problem of a reduced yield. In particular, the abundance of endogenous proteases in apoplast is a major obstacle to expression of the target protein in the apoplast. Furthermore, the site of the target protein recognized by a protease varies depending on the type of protein, and thus, it is difficult to predict the site.
[0036] To address these problems, a method of changing protein localization by using a protein sorting signal and a method of coexpressing a protease inhibitor in a plant have been used in the conventional art. However, the problem of degradation by a protease still remains unsolved for activin A.
[0037] Under such circumstances, the present inventors have found an unknown recognition site for proteases on activin A; and modified the amino acid sequence of a proregion (also referred to herein as "prodomain") of activin A, thereby successfully generating a modified activin A that is hard to be degraded by a protease other than a proprotein convertase (Furin) (has resistance to degradation by the protease). Thus, the present inventors have accomplished the present invention. The present invention provides a modified activin A that is hard to be degraded by the protease, by modifying the amino acid sequence of the proregion of activin A. The present inventors have found that activin A becomes hard to be degraded by the protease by modifying an amino acid sequence of a proregion of the activin A without modifying an amino acid sequence of a mature region of the activin A that is physiologically active.
[0038] The modified activin A of the present invention allows for improving the yield of activin A in producing the activin A in the presence of the protease other than a proprotein convertase (Furin) (for example, in planta), compared to an unmodified wild type activin A. In this respect, the modified activin A of the present invention is highly useful.
2. Activin A and Modified Form Thereof
[0039] Activin A is a cytokine belonging to the TGF.beta. (transforming growth factor-.beta.) superfamily. Activin A is a protein that is involved in development and differentiation as an erythroid differentiation factor and a mesoderm-inducing factor and regulates diverse functions in various cells.
[0040] The present invention provides activin A that is hard to be degraded by a protease other than a proprotein convertase (Furin) (has resistance to degradation by the above protease), by modifying the amino acid sequence of a proregion of activin A. Such activin A allows for improving the yield of activin A in producing the same in the presence of the protease, compared to a wild type activin A that is unmodified.
[0041] In the present invention, "activin A" refers to both or either one of pro-activin A and mature activin A. As the term "activin A" is used in the present invention, activin A may be a mixture of pro-activin A and mature activin A or may be either protein. Pro-activin A is a precursor of mature activin A and is a protein comprising a proregion, a recognition region for a proprotein converting enzyme, and a mature region (FIG. 1).
[0042] In the present invention, examples of the amino acid sequence of pro-activin A include, but not limited to, an amino acid sequence set forth in SEQ ID NO: 1 (human), SEQ ID NO: 2 (mouse), and SEQ ID NO: 3 (rat). In the present invention, examples of the amino acid sequence of the proregion include, but not limited to, an amino acid sequence set forth in SEQ ID NO: 4 (human), SEQ ID NO: 5 (mouse), and SEQ ID NO: 6 (rat). The amino acid sequence of the recognition region for a proprotein converting enzyme is an amino acid sequence set forth in SEQ ID NO: 7, and the amino acid sequence of the mature region (mature activin A) is an amino acid sequence set forth in SEQ ID NO: 8. Those skilled in the art can readily obtain these amino acid sequences and conformational information from known databases such as GenBank and UniProt Protein Data Bank (PDB). For example, human pro-activin A has GenBank accession number NP_002183.1 and PDB accession number 5HLY.
[0043] Mature activin A is a protein comprising a mature region, the protein being generated when pro-activin A is cleaved by a proprotein converting enzyme (Furin). Mature activin A is a homodimer formed by linking 116-amino acid residue .beta.A chains by a disulfide bond.
[0044] In the present invention, the type of animals from which activin A is derived is not limited. Examples of the animals include a human, a mouse, a rat, a guinea pig, a rabbit, a cat, a dog, a pig, a monkey, and a cow. Preferably, the animal is a human. The amino acid sequences of the mature region of activin A have 100% homology among humans, mice, rats, cats, pigs, and cows.
[0045] The present invention provides activin A that has resistance to degradation by a protease other than a proprotein convertase (Furin). In the present invention, a "protease other than a proprotein convertase (Furin)" is not limited, as long as the protease has the activity of degrading (for example, cleaving) activin A. The protease in the present invention may be an endogenous protease comprised in a transformant of a host such as a plant, a non-human mammal and a cell derived therefrom, a bacterium, a yeast, and a fungus, or may be an exogenous protease, which is found outside the transformant. The protease is preferably an endogenous plant protease or an exogenous plant protease, and more preferably an endogenous plant protease. Specifically, there are abundant proteases having various spectra (ranges) of specificities in a plant body (for example, apoplast) (Pillary P. et al., Bioengineered, 5: 1, 15-20, 2014), and thus, the endogenous plant protease is not limited, as long as the protease has the activity of degrading activin A.
[0046] Those skilled in the art can determine whether a specific protease has the activity of degrading activin A by using a known method such as SDS-PAGE and Western blotting.
[0047] In the present invention, the phrase "have resistance to degradation by a protease other than a proprotein convertase (Furin)" means that, in particular, when pro-activin A was expressed in a transformant, extracted, and purified as necessary and then the resulting protein was analyzed, the percentage of pro-activin A content relative to the total resulting protein is usually 70% or more, preferably 80% or more, and more preferably 84% or more. This phrase does not necessarily mean that the percentage of the pro-activin A content has to be 100% (not necessarily mean that degradation has to be suppressed by 100%). The amount or percentage of pro-activin A content can be measured by Western blotting as described in Examples. Thus, those skilled in the art can determine whether the modified activin A has resistance to degradation by a protease other than a proprotein convertase (Furin) by measuring the amount or percentage of the pro-activin A content relative to the total protein by Western blotting.
[0048] In the present invention, "protease other than a proprotein convertase (Furin)" may be expressed as "protease (excluding a proprotein convertase (Furin)."
[0049] One embodiment of the present invention is activin A comprising a modified proregion. A modified proregion is a proregion in which a sequence expected as a loop region is modified, and is preferably a proregion in which, among loop regions in the proregion, at least one sequence expected as a loop region has been deleted. Herein, the loop region is a part having a loop structure, and more specifically, a part from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6.
[0050] In the present invention, the amino acid sequence of the modified proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and/or added in an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 (activin A comprising a modified proregion is also referred to herein as "modified activin A").
[0051] The present invention also provides activin A comprising a modified proregion, wherein the amino acid sequence of the modified proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and/or added in an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6, and the activin A has resistance to degradation by a protease other than a proprotein convertase (Furin).
[0052] Examples of the amino acid sequence of the modified proregion include, but not limited to, the following amino acid sequences (a) to (d):
[0053] (a) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is deleted;
[0054] (b) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a spacer sequence consisting of 1 to 10 amino acids;
[0055] (c) an amino acid sequence in which at least one of amino acids from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a different amino acid; and
[0056] (d) an amino acid sequence in which an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 has at least one amino acid inserted therein.
[0057] Furthermore, examples of the amino acid sequence of the modified proregion include, but not limited to, amino acid sequences which are the following amino acid sequences (a) to (d) and wherein activin A comprising the amino acid sequence has resistance to degradation by a protease other than a proprotein convertase (Furin),
[0058] (a) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is deleted;
[0059] (b) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a spacer sequence consisting of 1 to 10 amino acids;
[0060] (c) an amino acid sequence in which at least one of amino acids from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a different amino acid; and
[0061] (d) an amino acid sequence in which an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 has at least one amino acid inserted therein.
[0062] The above described amino acid sequence (a) is the amino acid sequence set forth in any of SEQ ID NO: 9 (human), SEQ ID NO: 10 (mouse), and SEQ ID NO: 11 (rat).
[0063] In the amino acid sequence (b), a "spacer sequence" is an amino acid sequence consisting of 1 to 10 amino acids. The amino acid constituting the spacer sequence is at least one selected from glycine, alanine, and serine. Specifically, the spacer sequence may be an amino acid sequence composed of only one type of amino acid, such as only glycine or only alanine; or may be an amino acid sequence composed of two or more types of amino acids, for example: glycine and alanine; glycine and alanine and leucine. The number of amino acids in the spacer sequence is 1 to 10, preferably 1 to 5, more preferably 1 to 4, and even more preferably 1 to 3. More specifically, examples of the spacer sequence include -G-, -GG-, -GGG-, and -GGGG-. In the present invention, when a spacer consists of one amino acid residue, "spacer sequence" may also be referred to as "spacer residue."
[0064] Those skilled in the art can search for the type and number of amino acid candidates as well as amino acid sequence candidates that constitute the spacer sequence by using molecular visualization software described below. Furthermore, in the present invention, a substructure can be inserted instead of the spacer sequence, provided that the substructure is registered in PDB, has ends whose relative coordinates correspond to the coordinates of an insertion site on activin A, can form a peptide bond, and does not cause a steric hindrance after insertion.
[0065] Examples of the above described amino acid sequence (b) include, but not limited to, an amino acid sequence set forth in any of SEQ ID NO: 12 (human), SEQ ID NO: 13 (mouse), and SEQ ID NO: 14 (rat) that comprises one glycine as the spacer sequence; an amino acid sequence set forth in any of SEQ ID NO: 15 (human), SEQ ID NO: 16 (mouse), and SEQ ID NO: 17 (rat) that comprises two glycines as the spacer sequence; and an amino acid sequence set forth in any of SEQ ID NO: 18 (human), SEQ ID NO: 19 (mouse), and SEQ ID NO: 20 (rat) that comprises three glycines as the spacer sequence.
[0066] In the above amino acid sequence (c), a "different amino acid" is not limited as long as the amino acid does not cause a steric hindrance in activin A. Examples thereof include alanine, serine, glycine, valine, leucine, and isoleucine.
[0067] In the above amino acid sequence (d), the amino acid to be inserted is not limited as long as the amino acid does not cause a steric hindrance in activin A. Examples thereof include alanine, serine, glycine, valine, leucine, and isoleucine.
[0068] In the present invention, in addition to the above described amino acid sequence set forth in any of SEQ ID NOs: 9 to 20, the following amino acid sequences can be used as an amino acid sequence of the modified proregion:
[0069] (f) an amino acid sequence in which one or several amino acids are deleted, substituted or added in an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence has resistance to degradation by a protease other than a proprotein convertase (Furin); and
[0070] (g) an amino acid sequence having 80% or more homology with an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence has resistance to degradation by a protease other than a proprotein convertase (Furin).
[0071] In the above amino acid sequence (f), examples of an amino acid sequence in which one or several amino acids are deleted, substituted or added in set forth in any of SEQ ID NOs: 9 to 20 include the following amino acid sequences:
[0072] (i) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids of an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20 is deleted;
[0073] (ii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids of an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20 is substituted with a different amino acid;
[0074] (iii) an amino acid sequence in which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids are added to an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20; and
[0075] (iv) an amino acid sequence mutated by a combination of the above described (i) to (iii).
[0076] In the above amino acid sequence (g), examples of the amino acid sequence having 80% or more homology with an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20 include an amino acid sequence having 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20.
[0077] Those skilled in the art can readily examine homology among amino acid sequences by using a known database such as FASTA or BLAST.
[0078] In the present invention, "resistance to degradation by a protease other than a proprotein convertase (Furin)" means the property that makes activin A hard to be degraded by a protease other than a proprotein convertase (Furin). Furthermore, as described above, the phrase "have resistance to degradation by a protease other than a proprotein convertase (Furin)" in the present invention means that, in particular, when pro-activin A was expressed in a transformant, extracted, and purified as necessary and then the resulting protein was analyzed, the percentage of pro-activin A content relative to the total resulting protein is usually 70% or more, preferably 80% or more, and more preferably 84% or more. This phrase does not necessarily mean that the percentage of the pro-activin A content has to be 100% (not necessarily mean that degradation has to be suppressed by 100%). Those skill in the art can determine whether a modified activin A has resistance to degradation by a protease other than a proprotein convertase (Furin) by measuring the amount or percentage of the pro-activin A content relative to the total protein by Western blotting, as described in Examples.
[0079] The present invention also provides activin A comprising a modified proregion, wherein the amino acid sequence of the modified proregion comprises an amino acid sequence in which at least one amino acid has been deleted, substituted and/or added in an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6, and wherein the activin A has the activity of inducing differentiation into an erythroblast when cleaved by a proprotein converting enzyme.
[0080] Examples of the amino acid sequence of the modified proregion include, but not limited to, the below described amino acid sequences (a) to (g). Examples of the activin A comprising a modified proregion include, but not limited to, the activin A comprising any of the below described amino acid sequences (a) to (g) and having the activity of inducing differentiation into an erythroblast when cleaved by a proprotein converting enzyme.
[0081] (a) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is deleted;
[0082] (b) an amino acid sequence in which an amino acid sequence from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a spacer sequence consisting of 1 to 10 amino acids;
[0083] (c) an amino acid sequence in which at least one of amino acids from position 182 to 199 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 is substituted with a different amino acid;
[0084] (d) an amino acid sequence in which an amino acid sequence from position 180 to 201 of an amino acid sequence set forth in any of SEQ ID NOs: 4 to 6 has at least one amino acid inserted therein;
[0085] (e) an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20;
[0086] (f) an amino acid sequence in which one or several amino acids are deleted, substituted or added in an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20; and
[0087] (g) an amino acid sequence having 80% or more homology with an amino acid sequence set forth in any of SEQ ID NOs: 9 to 20.
[0088] In the present invention, "has the activity of inducing differentiation into an erythroblast when cleaved by a proprotein converting enzyme" means that activin A resulting from cleavage by a proprotein converting enzyme (a mixture of pro-activin A and mature activin A or mature activin A) has the activity of inducing differentiation of any erythroblastic cultured cell (for example, a F5-5 cell and a K562 cell) into an erythroblast.
[0089] The activity of inducing differentiation into an erythroblast can be evaluated by a known method. An exemplary method comprises adding a test polypeptide (test activin A) to the culture solution of erythroblastic cultured cells, staining the cells after cultivation with aminoethylcarbazole (AEC) or the like, and measuring the ED.sub.50 value of the test activin A to determine the differentiation rate.
[0090] In the present invention, examples of the amino acid sequence of activin A comprising a modified proregion include, but not limited to, amino acid sequences of activin A comprising each of the amino acid sequences set forth in SEQ ID NOs: 9 to 20 (amino acid sequences set forth in SEQ ID NOs: 21 to 32).
[0091] The conformation of activin A with modification (deletion, substitution, addition, or a combination thereof) can be analyzed and simulated by using molecular visualization software that displays the structure of a protein in a 3D image based on data in PDB or the like. Examples of such software include Chimera and PyMOL. For example, the above amino acid sequence (a), in which the amino acid sequence from position 182 to 199 has been deleted and no spacer sequence has been inserted, can be subjected to simulation based on an in silico analysis using Chimera to find out whether a steric hindrance occurs. For the above amino acid sequence (b), the above software can be used to search for the type and number of amino acids or amino acid sequence candidates that constitute the spacer sequence. Similarly, the above amino acid sequences (c) and (d) can be subjected to simulation to find out whether a steric hindrance or the like occurs when substitution or insertion by a different amino acid was introduced in the amino acid sequence from position 182 to 199. Thus, those skilled in the art can select appropriately an amino acid residue that causes no steric hindrance when being deleted, substituted and/or added in activin A by using such molecular visualization software.
[0092] Furthermore, the chemical property or physical property of amino acids (polar/non-polar, acidic/basic, hydrophilic/hydrophobic, side chain size, and the like) is known. Those skilled in the art can appropriately select an amino acid to be deleted, substituted, and/or added on the basis of these properties. Substitution between amino acids that have similar structures or properties is predicted to be hard to substantially inhibit the biological activity of a polypeptide.
[0093] 1) Polar amino acids: lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tyrosine, and cysteine;
[0094] 2) Nonpolar amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, and proline;
[0095] 3) Acidic amino acids: aspartic acid and glutamic acid;
[0096] 4) Basic amino acids: lysine, arginine, and histidine;
[0097] 5) Neutral amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, methionine, cysteine, and proline;
[0098] 6) Aliphatic amino acids: glycine, alanine, valine, leucine, and isoleucine;
[0099] 7) Aromatic amino acids: phenylalanine, tyrosine, and tryptophan;
[0100] 8) Amino acids having a hydroxyl group: serine and threonine;
[0101] 9) Amino acids having an amido group: asparagine and glutamine;
[0102] 10) Sulfur-containing amino acids: methionine and cysteine;
[0103] 11) Hydrophobic amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, methionine, cysteine, and proline (it is noted that glycine, tyrosine, tryptophan, and cysteine may be classified as hydrophilic amino acids);
[0104] 12) Hydrophilic amino acids: lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine, and threonine.
3. Polynucleotide, Vector, and Transformant
(1) Polynucleotide
[0105] The nucleotide sequence of a polynucleotide of the present invention is not limited as long as the sequence is DNA or RNA encoding a modified activin A of the present invention. Those skilled in the art can optimize codons for the polynucleotide depending on the type of host in which activin A is expressed. This optimization can improve the amount of the modified activin A expressed in the host. Examples of the polynucleotide encoding the modified activin A of the present invention include, but not limited to, a polynucleotide comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 33 to 36 (all thereof are human nucleotide sequences).
[0106] In addition to the polynucleotide comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 33 to 36, a polynucleotide described below can be used as the polynucleotide of the present invention. Such a polynucleotide is a polynucleotide that hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence set forth in any of SEQ ID NOs: 33 to 36 under stringent conditions and encodes activin A having resistance to degradation by a protease other than a proprotein convertase (Furin). In the present invention, a "stringent condition" may be either a low stringency condition, a moderate stringency condition, or a high stringency condition. A "low stringency condition" is, for example, the following condition: 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS, 50% formamide, and 32.degree. C. A "medium stringency condition" is, for example, the following condition: 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS, 50% formamide, and 42.degree. C. A "high stringency condition" is, for example, the following condition: 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS, 50% formamide, and 50.degree. C. For a detailed procedure of a hybridization method, one can refer to "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)), and the like.
[0107] As the polynucleotide of the present invention, a polynucleotide that has 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the nucleotide sequence set forth in any of SEQ ID NOs: 33 to 36, and encodes activin A having resistance to degradation by a protease other than a proprotein convertase (Furin) can also be used.
[0108] Methods for introducing a mutation into a polynucleotide are known. For example, those skilled in the art can introduce a mutation into the polynucleotide of the present invention by using a known method including a site-directed mutagenesis strategy such as the Kunkel method and the Gapped duplex method, the overlap extension PCR method and the QuikChange method. The amino acid sequence of activin A can be modified by introducing a mutation into the polynucleotide of the present invention.
[0109] In the present invention, an affinity tag can be added to activin A for affinity-purifying activin A. Examples of usable affinity tags include, but not limited to, a histidine tag (an amino acid sequence consisting of contiguous histidine residues), a GST tag, a FLAG tag, and a c-myc tag. Methods for adding a histidine tag are known and those skilled in the art can readily add a histidine tag to the N-terminus of the activin A of the present invention by using a known method. One example of the amino acid sequences of activin A with a histidine tag added to the N-terminus thereof is set forth in SEQ ID NO: 37.
[0110] In the present invention, a signal sequence can also be added to activin A. Examples of the signal sequence include a KDEL signal. Methods for adding a signal sequence are known. A sequence optimized for expression thereof in a plant cell can be used.
(2) Vector
[0111] In the present invention, a "vector" is not limited as long as the vector comprises the polynucleotide of the present invention described in the above (1). For example, a plasmid vector, a viral vector, an Agrobacterium bacterium, or the like can be used. When a plant is used as a host, for example, a plant viral vector, an Agrobacterium bacterium, or the like can be used as the vector. More specifically, for example, a TMV-based vector, a PVX-based vector, a CPMV-based vector, a CMV-based vector, a PPV-based vector, an AIMV-based vector, a ZYMV-based vector, or the like can be used. The vector used in the present invention may be either a vector for transient expression or a vector for constitutive expression. Those skilled in the art can appropriately select a vector to be used depending on the type of host into which the vector is introduced and the purpose.
[0112] In the vector of the present invention, an additional sequence may be linked to the polynucleotide of the present invention, as desired. Examples of the additional sequence include a cis element such as an enhancer, a splicing signal, a poly(A) addition signal, a ribosome binding sequence (SD sequence), a selective marker gene, and a reporter gene. Examples of the selective marker gene include a dihydrofolate reductase gene, an ampicillin-resistant gene, and a neomycin-resistant gene. Examples of the reporter gene include genes for green fluorescent protein (GFP) or variants thereof (fluorescent proteins such as EGFP, BFP, and YFP), luciferase, alkaline phosphatase, and LacZ.
(3) Transformant
[0113] In the present invention, a transformant can be obtained by introducing the vector described in the above (2) into a host. In the present invention, a "transformant" is a non-human transformant. In the present invention, a "host" is an organism into which the vector of the present invention is introduced and in which a modified activin A of interest is expressed. The host is not limited as long as the host expresses the modified activin A of the present invention. For example, a plant, a non-human mammal and a cell derived therefrom, a bacterium, a yeast, a fungus, or the like can be used, and a plant is preferred. Examples of the plant include a Nicotiana plant, a moss plant (for example, Physcomitrella patens (P. patens)), a potato (for example, S. tuberosum), a gramineous plant (for example, O. sativa), a cruciferous plant, and lettuce; and a Nicotiana plant is preferred. Examples of the Nicotiana plant include, but not limited to, Nicotiana benthamiana (N. benthamiana), Nicotiana tabacum (N. tabacum), and Nicotiana excelsior (N. excelsior). Examples of the non-human mammal include, but not limited to, a mouse, a rat, a guinea pig, a rabbit, a cat, a dog, a pig, a monkey, and a cow, and a cell derived therefrom. Examples of the bacterium include, but not limited to, E. coli.
[0114] Introduction of a vector into a host can be performed by using a known method. Examples of the known gene introduction method include a method using a viral vector, an agroinfection method, a calcium phosphate method, a microinjection method, a particle gun method, a DEAE-dextran method, electroporation, and a cationic lipid-mediated method. When the host is a plant, the method using a viral plant vector and the agroinfection method are preferred. The vectors that can be used in these methods are as described in the above (2) as "a vector when a plant is used as a host."
[0115] In the present invention, when a plant is used as a host, the plant is cultivated before transformation. A method for cultivating plants is as described below.
[0116] First, seeds are sown on a seedling tray supplemented with a fertilizer, and the plant after sowing is grown for several days in an artificial climate chamber with a controlled light cycle. When a liquid fertilizer is used as the fertilizer, a urethane mat for hydroponics can be impregnated with the liquid fertilizer, and then placed in the seedling tray.
[0117] Then, the grown plant body is transplanted to a panel for cultivation (first period), the panel after transplantation is placed in the artificial climate chamber, and cultivation is performed for several days by, for example, a deep flow technique (DFT system).
[0118] Subsequently, the plant body is taken out from the panel for cultivation (first period) and planted on a panel for cultivation (second period). The panel for cultivation (second period) after transplantation is placed in the artificial climate chamber and cultivation is performed for several days by the DFT system to obtain the plant body.
[0119] In the above described method, a liquid fertilizer can be used as the fertilizer, but the fertilizer is not limited thereto. When a liquid fertilizer is used, the urethane mat for hydroponics can be impregnated with the liquid fertilizer, and then placed in the seedling tray.
[0120] In the present invention, liquid fertilizers are not limited, and commercially available liquid fertilizers can be used in appropriate combinations. The liquid fertilizer can be dissolved in dechlorinated water. Furthermore, the electric conductivity and pH of the liquid fertilizer can be adjusted before use. Those skilled in the art can adjust these by a known method.
[0121] In the present invention, examples of the environmental condition include, but not limited to, a condition with a temperature of 10 to 40.degree. C. (for example, 28.degree. C.), a relative humidity of 60 to 80%, a CO.sub.2 concentration of 300 to 5000 ppm (for example, 400 ppm or 500 ppm), and the number of days of cultivation for the first period of 0 to 35 days (for example, 9 days) and that for the second period of 0 to 35 days (for example, 7 days). Those skilled in the art can adjust these parameters as appropriate depending on, for example, the growth status of the plant.
[0122] In the present invention, as hydroponic techniques, the deep flow technique (DFT system) and a nutrient film technique (NFT system) can be mainly used.
[0123] As described above, when the host is a plant, the method using a plant viral vector and the agroinfection method can be used. These methods are well known to those skilled in the art. By way of example, a brief description of the agroinfection method is as follows.
[0124] First, the vector of the present invention described in the above (2) is introduced into an Agrobacterium bacterium by electroporation, thereby transforming the Agrobacterium bacterium. Examples of Agrobacterium bacterium that can be used in the present invention include, but not limited to, the GV3101 strain, the LBA4404 strain, the EHA101 strain, the EHA105 strain, and the AGL1 strain.
[0125] Then, plant leaves or the like are infected with the transformed Agrobacterium bacterium. Examples of methods for infecting a plant with an Agrobacterium bacterium include a vacuum infiltration method, a syringe infiltration method, a leaf disc method, and a foliar application method. An exemplary procedure for using the vacuum infiltration method is as follows. First, the cultivated plant is turned upside down and immersed in the bacterial suspension of Agrobacterium bacteria in a beaker such that all the leaves are completely immersed in the suspension. Subsequently, the beaker is placed in a vacuum desiccator and allowed to stand for several minutes (for example, one minute) under reduced pressure. Then, the valve is fully opened to restore pressure. After completing pressure restoration, the plant is returned to an upright position and planted in the artificial climate chamber. After infection, the plant is cultivated for 1 to 14 days (for example, 6 days) in the artificial climate chamber by, for example, the DFT system. The environmental condition is similar to that described above, and those skilled in the art can adjust these parameters as appropriate depending on, for example, the growth status of the plant.
[0126] In this way, a transformant of the plant can be generated. In infecting a plant with the Agrobacterium bacterium, the plant may be coinfected with multiple types of Agrobacterium bacteria, each comprising a different vector. In this case, Agrobacterium bacteria comprising a vector other than the vector of the present invention may be used in combination. Examples of such vector include a vector comprising a signal peptide of a barley .alpha.-amylase or a rice .alpha.-amylase, and a PhiC31 integrase expression vector.
[0127] Transformants from other hosts can also be generated readily by those skilled in the art by a known method described in, for example, "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)).
4. Method for Producing Activin A
[0128] In the present invention, activin A can be produced by recovering activin A from the transformant of the present invention described in the above section 3. Furthermore, in the method of the present invention, a mature region of activin A in which a proregion is removed (mature activin A) can be obtained by purifying the recovered activin A (pro-activin A) and treating the purified pro-activin A with a proprotein convertase (for example, Furin).
[0129] The type of transformant used in the method for producing activin A is not limited. By way of example, when the transformant is a plant, the method for producing activin A is as described below.
[0130] First, leaves are collected from the transformed plant body that was cultivated for 1 to 14 days (for example, 6 days) after infection with Agrobacterium bacterium, and activin A (pro-activin A) is extracted by using an extraction buffer. The quantity of leaves of the plant body to be collected depends on the type of plant body. The leaves can be frozen and stored at -80.degree. C. before extraction. Examples of the extraction buffer include, but not limited to, phosphate buffer, Tris buffer, and acetate buffer. The pH is usually adjusted to a range between pH 2 and 11, including the range in which the above described buffers work properly.
[0131] Next, activin A comprised in the extract is purified. Purification can be performed by using conventional methods alone or in appropriate combinations. The conventional methods are, for example, aqueous two-phase partition, ammonium sulfate fractionation, affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, or reverse-phase chromatography.
[0132] Conventional methods can be used to confirm that the resulting purified substance is the target protein, activin A. Examples of the conventional methods include SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting, enzyme immunoassay (ELISA), and mass spectrometry.
[0133] In this way, purified activin A (pro-activin A) can be obtained.
[0134] Furthermore, the mature region of activin A in which a proregion is removed (mature activin A) can be obtained by treating the purified pro-activin A with a proprotein convertase (Furin). A mixture of the proregion and the mature activin A may also be used for experiments.
[0135] The activity of the obtained mature activin A (or the mixture of the proregion and the mature activin A) can be evaluated, for example by cultivating a F5-5 cell line (Friend erythroblastic leukemia cell line) in a culture medium supplemented with the mature activin A, then staining the cells with AEC (aminoethylcarbazole), and measuring the ED.sub.50 value (indicating the concentration of mature activin A at which the differentiation rate is 50%) of the mature activin A for the differentiation rate.
5. Composition Comprising Activin A
[0136] The present invention can provide a composition comprising a modified activin A of the present invention. The composition of the present invention may comprise known additives such as saline, buffer, and an excipient, in addition to the modified activin A.
[0137] Hereinbelow, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.
Example 1
1. Designing Modified Activin A
[0138] In this example, a sequence from which a specific sequence of a proregion was removed was designed.
[0139] When pro-activin A, which was transiently expressed by a plant, was extracted and then subjected to Western blotting, a plurality of unexpected degradation products was found. Thus, a plurality of sequences to induce site-specific mutations was designed and mutations were induced by using QuikChange (Agilent Technologies, Inc.). The mutated protein was overexpressed and examined in the same way as the above described original sequence. As a result, it was confirmed that the degradation was reduced in more than one sequences. The sequences were a sequence from which Pro182-Gly192 was removed. The Pro182-Gly192 was predicted to be a loop region of the prodomain. PDB 5HLY was used to adjust the dihedral angle between H181 and E200 and these residues were linked by a peptide bond. As a result, it was found not to cause a steric hindrance, and thus, this protein was designed as Mut3-1. Furthermore, an appropriate linker sequence was introduced between the above H181 and E200. A sequence into which (Gly)n (n=1 to 4) was introduced was designed to perform expression screening. Based on SuperLooper (http://bioinf-applied.charite.de/superlooper/description.php), the above linkers (Gly)n (n=2 or 3) that have the relative position coordinates of the residues, H181 and E200 for linker introduction were extracted from the registered PDB structure information (3DZM, 2VK3, and 5AA5 having GG, and 4GNO having GGG). Thus, sequences of Mut3-2 (n=1), Mut3-3 (n=2), and Mut3-4 (n=3) were designed.
Example 2
Production of Modified Activin A
[0140] On the basis of the information of the modified activin A designed in Example 1, the modified activin A was produced by a below described method.
1. Preparation of Vector
[0141] A vector for expressing pro-activin A in which a histidine tag (HHHHHH) was added to the N-terminus thereof was generated as described below. A gene encoding pro-activin A in which a histidine tag was added to the N-terminus thereof was inserted into a Bsa I site of a tobacco mosaic virus (TMV) 3'-provector (pICH31070, NOMAD Bioscience GmbH) by Golden Gate cloning method (Engler et al., 2008), thereby generating a pN-His-Pro-activin.
[0142] Next, a TMV 3'-provector that expresses a gene encoding a modified pro-activin A was generated as described below. PCR was carried out by using QuikChange Lightning Kit (Agilent Technologies, Inc.) with the pN-His-Pro-activin as a template and the following primers for mutation introduction, thereby generating a pMut3-1.
TABLE-US-00001 (SEQ ID NO: 38) 5'-CCAACAGCAGAAGCACGAGAGATCTGAGTTGC-3' (SEQ ID NO: 39) 5'-GCAACTCAGATCTCTCGTGCTTCTGCTGTTGG-3
2. Generation of Transformant
(1) Cultivation of Host Plant
[0143] In this example, Nicotiana benthamiana, which is a Nicotiana plant, was used as a host plant.
(1-1) Sowing
[0144] A urethane mat for hydroponics (Ematsu Kasei; (W)587.5 mm.times.(D)282 mm.times.(H)28 mm; 12.times.2 blocks; hole diameter (.PHI.): 9 mm) was impregnated with a liquid fertilizer for sowing (0.78 g/L Otsuka House No. S1 (Otsuka AgriTechno Co., Ltd.) and 0.25 g/L Otsuka House No. 2 (Otsuka AgriTechno Co., Ltd.); pH 5.0) and placed in a seedling tray ((W)600 mm.times.(D)300 mm.times.(H)300 mm). Then, seeds of Nicotiana benthamiana were sown on the mat.
(1-2) Raising Seedlings
[0145] The plant after sowing was grown in an artificial climate chamber (NC-410HC) (Nippon Medical & Chemical Instruments Co., Ltd.) at a room temperature of 28.degree. C. with a light cycle of 16 hours of light and 8 hours of darkness for 12 days.
(1-3) Cultivation (First Period)
[0146] The urethane mat used for raising seedlings was divided into each block and transplanted to a panel for cultivation (first period) ((W)600 mm.times.(D)300 mm, 30 holes). The panel for cultivation (first period) after transplantation was placed in the artificial climate chamber (LH-410SP) (Nippon Medical & Chemical Instruments Co., Ltd.) and cultivation was performed for 9 days by a deep flow technique (DFT system). The environmental conditions and the liquid fertilizer conditions were controlled as follows.
Environmental Conditions
Temperature: 28.degree. C.
[0147] Relative humidity: 40 to 60% CO.sub.2 concentration: 400 ppm Lighting: average photosynthetic photon flux density (PPFD): 140 .mu.molm.sup.-2sec.sup.-1, 24 hours continuous lighting, a three band fluorescent lamp "Rupika Line" (Mitsubishi Electric Corporation)
Liquid Fertilizer Conditions
[0148] Fertilizer liquid A (150 g/L Otsuka House No. S1 and 2.5 g/L Otsuka House No. 5 (Otsuka AgriTechno Co., Ltd.)) and fertilizer liquid B (100 g/L Otsuka House No. 2) were dissolved individually in dechlorinated water and these solutions were mixed in equal proportions. The resulting mixture was used as the liquid fertilizer. A pH adjuster "Down" (Otsuka AgriTechno Co., Ltd.) and 4% aqueous KOH solution were used to adjust pH. The electrical conductivity (EC) and the pH of the liquid fertilizer were adjusted to an EC of 2.3 mS/cm and pH 6.0, respectively, by using "Easy Fertilizer Controller RAKU-RAKU 3" (CEM Corporation).
(1-4) Cultivation (Second Period)
[0149] The plant body was taken out from the panel for cultivation (first period) and planted on a panel for cultivation (second period) ((W)600 mm.times.(D)300 mm, 6 holes). The panel for cultivation (second period) after transplantation was placed in the artificial climate chamber (LH-410SP) (Nippon Medical & Chemical Instruments Co., Ltd.) and cultivation was performed for 7 days by the DFT system (for 28 days after sowing). Environmental conditions were controlled as described below.
Environmental Conditions
Temperature: 28.degree. C.
[0150] Relative humidity: 60 to 80% CO.sub.2 concentration: 500 ppm Lighting: average photosynthetic photon flux density (PPFD): 140 molm.sup.-2sec.sup.-1, 24 hours continuous lighting, a three band fluorescent lamp "Rupika Line" (Mitsubishi Electric Corporation)
(2) Generation of Transformant
(2-1) Infection by Vacuum Infiltration
[0151] The vectors of the present invention generated in the above "1." were individually introduced into an Agrobacterium GV3101 strain by electroporation. Then, agroinfiltration was used to coinfect Nicotiana benthamiana with each of the obtained GV3101 strains: a GV3101 strain carrying a 5'-provector comprising a signal peptide of rice .alpha.-amylase (pICH20155, NOMAD Bioscience GmbH) and a GV3101 strain carrying a PhiC31 integrase expression vector (pICH14011, NOMAD Bioscience GmbH).
[0152] Specifically, the Nicotiana benthamiana obtained on Day 28 after sowing in the above section "2.(1)" was turned upside down and immersed in the bacterial suspension of Agrobacterium bacteria in a beaker such that all the leaves were completely submerged in the suspension.
[0153] Subsequently, the beaker was placed in a vacuum desiccator (FV-3P) (Tokyo Glass Kikai Co., Ltd.) and allowed to stand for one minute under reduced pressure at -0.09 MPa. Then, the valve was fully opened to restore pressure.
[0154] After completing pressure restoration, the plant was returned to an upright position and planted in the artificial climate chamber (LH-410SP) (Nippon Medical & Chemical Instruments Co., Ltd.).
(2-2) Cultivation of Infected Leaves (Expression Step)
[0155] Cultivation after infection was performed by using the artificial climate chamber (LH-410SP) (Nippon Medical & Chemical Instruments Co., Ltd.). Cultivation was performed for six days by the DFT system. Environmental conditions were controlled as described below.
Environmental Conditions
Temperature: 28.degree. C.
[0156] Relative humidity: 60 to 80% CO.sub.2 concentration: 500 ppm Lighting: average photosynthetic photon flux density (PPFD): 140 .mu.molm.sup.-2sec.sup.-1, 24 hours continuous lighting, a three band fluorescent lamp "Rupika Line" (Mitsubishi Electric Corporation)
3. Production of Modified Pro-Activin A
(1) Extraction
[0157] Tobacco leaves were harvested from the transformed Nicotiana benthamiana six days after infection. The leaves were frozen and stored at -80.degree. C. until extraction.
[0158] A buffer comprising 0.1 M sodium phosphate, 0.5 M arginine, and 5 mM sodium sulfite, pH 8.0 (or a phosphate buffer) was used as an extraction buffer.
(2) Purification
(2-1) Purification by Ammonium Sulfate Fraction
[0159] Ammonium sulfate was added to the supernatant collected by the above described method to produce a 35% saturated ammonium sulfate solution. After stirring the mixture at room temperature for one hour, the mixture was centrifuged at 15,000.times.g for 15 minutes at room temperature to collect the supernatant of 35% ammonium sulfate fractionation. Subsequently, ammonium sulfate was added thereto to produce a 60% saturated ammonium sulfate solution and stirred at room temperature for one hour. This solution was centrifuged at 15,000.times.g for 15 minutes to collect precipitates of the 60 to 90% ammonium sulfate fractionation.
(2-2) Purification by Affinity Chromatography
[0160] To 50 g of precipitates of the 60 to 90% ammonium sulfate fractionation from activin A-expressing tobacco leaves, 10 mL of equilibration solution for histidine (His)-tag affinity purification (20 mM HEPES, 150 mM NaCl, 10% (w/v) glycerol, pH 8.0) was added, and the precipitate was dissolved. This solution was passed through a HisTrap HP 1 mL column (GE Healthcare) equilibrated with the equilibration solution for His-tag affinity purification at a speed to achieve retention time of 3 minutes. Then, the column was washed with a washing solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 20 mM imidazole, 10% (w/v) glycerol, pH 8.0). Lastly, an elution solution for His-tag affinity purification (20 mM HEPES, 150 mM NaCl, 200 mM imidazole, 10% (w/v) glycerol, pH 8.0) was passed through the column and the elution peak was collected to obtain a modified pro-activin A.
4. Production of Mature Activin A
[0161] A proprotein convertase (Furin) was added to the modified pro-activin A obtained by the above described method (Mut3-1, Mut3-2, Mut3-3, and Mut3-4 (comprising the amino acid sequences of SEQ ID NOs: 9, 12, 15, and 18, respectively)) under conditions of 25 mM Tris-HCl, pH 8.0, 10% glycerol, and 1 mM CaCl.sub.2 to obtain a mature activin A.
[0162] In this example, activin A in which an amino acid sequence of a proregion was modified (a modified pro-activin A and a mature activin A) was successfully produced.
Example 3
1. Functional Analysis of Modified Pro-Activin A
[0163] The purified modified pro-activin A obtained in "3. Production of Modified Pro-activin A" of Example 2 was analyzed by Western blotting to determine how a plant endogenous protease degraded the protein. Specifically, Penta-His Antibody HRP Conjugate (QIAGEN) was used as an antibody and the samples comprising each modified pro-activin A were subjected to Western blotting. LumiGLO Reagent and Peroxide (Cell Signaling Technology, Inc.) was used as a luminescent reagent and was detected by ImageQuant LAS 500 (GE Healthcare). The obtained Western blotting image is shown in FIG. 2. Wild-type Activin A (N-His-Activin A in the figure) (SEQ ID NO: 37) was used as a control. Densitometric analysis was also performed by using analysis software ImageJ and the results are shown in Table 1. In Table 1, "other polypeptides" are peptides resulting from degradation of the expressed proteins by the plant endogenous protease.
TABLE-US-00002 TABLE 1 N-His- Expressed protein Activin A Mut3-1 Mut3-2 Mut3-3 Mut3-4 Pro-activin A 63% 87% 84% 86% 89% Other polypeptides 37% 13% 16% 14% 11%
[0164] The results indicate that degradation of the modified activin A of the present invention by the plant endogenous protease was significantly suppressed compared to the nonmodified activin A (N-His-Activin A, which is the wild type activin A) (FIG. 2).
[0165] This example demonstrated that the modified activin A of the present invention had the property of being hard to be degraded by a protease (resistance to degradation by a protease). This example also demonstrated that the modified activin A of the present invention was extremely useful in producing activin A in the presence of a protease, which is present in a host for expression, in a purification step, in a reagent and/or culture medium to be used, and the like. The reason for this is that the modified activin A of the present invention allows for improving the amount of expression thereof and/or the yield thereof compared to the nonmodified activin A.
2. Evaluation of Activity of Mature Activin A
[0166] The activity of the mature activin A obtained in "4. Production of Mature Activin A" of Example 2 was evaluated. F5-5 cells cultured in an incubator (37.degree. C., 5% CO.sub.2) were seeded in a 96 well plate and cultured in the incubator after adding each mature activin A. Then, the differentiation rate of the cells was determined by staining the cells with AEC (aminoethylcarbazole, Tokyo Chemical Industry Co., Ltd.), according to the method described in Non Patent Literature 5 (Koretz K. et al., Histochemistry, 86: 5, 471-8, 1987). Table 2 shows the ED.sub.50 value of the mature activin A derived from the wild type activin A (N-His-Activin A) and the mature activin A derived from the modified activin A (Mut3-1 to Mut3-4).
TABLE-US-00003 TABLE 2 Expressed protein ED.sub.50 ng/mL N-His-Activin A <5 Mut3-1 <5 Mut3-2 <5 Mut3-3 <5 Mut3-4 <5
[0167] As shown in Table 2, the ED.sub.50 values of the mature activin A derived from the modified activin A ("Mut3-1" to "Mut3-4" in Table 2) were all less than 5 ng/mL, which were comparable to those of the mature activin A derived from the wild-type activin A ("N-His-Activin A" in Table 2).
[0168] These results indicated that the mature activin A obtained by treating the modified activin A with Furin had an ability to induce differentiation of the F5-5 cell into an erythroblast, with the same level of the wild-type activin A, and thus, it had an activity as mature activin A with same level of the activity of the wild-type activin A.
INDUSTRIAL APPLICABILITY
[0169] Use of the modified activin A of the present invention allows for improving the yield of activin A in producing the same in the presence of a protease.
[Sequence Listing Free Text]
[0170] SEQ ID NOs: 9 to 32 and 37: synthetic peptides SEQ ID NOs: 33 to 36, 38, and 39: synthetic DNA
Sequence CWU
1
1
391426PRTHomo sapiens 1Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser
Cys Trp Ile1 5 10 15Ile
Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20
25 30Pro Asp Cys Pro Ser Cys Ala Leu
Ala Ala Leu Pro Lys Asp Val Pro 35 40
45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn
50 55 60Met Leu His Leu Lys Lys Arg Pro
Asp Val Thr Gln Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly
Lys Val Gly 85 90 95Glu
Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu
100 105 110Met Asn Glu Leu Met Glu Gln
Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120
125Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu
Gly 130 135 140Ser Asp Leu Ser Val Val
Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145 150
155 160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val
Thr Ile Arg Leu Phe 165 170
175Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala
180 185 190Glu Glu Val Gly Leu Lys
Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200
205Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro
Val Ser 210 215 220Ser Ser Ile Gln Arg
Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val225 230
235 240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser
Gly Ala Ser Leu Val Leu 245 250
255Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys
260 265 270Gly Gly Gly Glu Gly
Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser 275
280 285His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser
Glu Asp His Pro 290 295 300His Arg Arg
Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile305
310 315 320Cys Cys Lys Lys Gln Phe Phe
Val Ser Phe Lys Asp Ile Gly Trp Asn 325
330 335Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn
Tyr Cys Glu Gly 340 345 350Glu
Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe 355
360 365His Ser Thr Val Ile Asn His Tyr Arg
Met Arg Gly His Ser Pro Phe 370 375
380Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser385
390 395 400Met Leu Tyr Tyr
Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln 405
410 415Asn Met Ile Val Glu Glu Cys Gly Cys Ser
420 4252424PRTMus musculus 2Met Pro Leu Leu Trp
Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu
Gly His Gly Ser Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro
35 40 45Asn Ser Gln Pro Glu Met Val Glu
Ala Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn
Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile
Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu
115 120 125Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu
Lys145 150 155 160Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His Pro Gln
Gly Ser Leu Asp Thr Gly Asp Glu Ala 180 185
190Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu
Ser Glu 195 200 205Lys Val Val Asp
Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210
215 220Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser
Ser Leu Asp Val225 230 235
240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu
245 250 255Leu Gly Lys Lys Lys
Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260
265 270Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu
Gln Ser His Arg 275 280 285Pro Phe
Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 290
295 300Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys
Val Asn Ile Cys Cys305 310 315
320Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp
325 330 335Ile Ile Ala Pro
Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 340
345 350Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser
Leu Ser Phe His Ser 355 360 365Thr
Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 370
375 380Leu Lys Ser Cys Cys Val Pro Thr Lys Leu
Arg Pro Met Ser Met Leu385 390 395
400Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn
Met 405 410 415Ile Val Glu
Glu Cys Gly Cys Ser 4203424PRTRattus norvegicus 3Met Pro Leu
Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro Gly
Ser Glu Gly His Gly Ala Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro
35 40 45Asn Ser Gln Pro Glu Met Val
Glu Ala Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu
Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp
Ile Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu
115 120 125Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu
Lys145 150 155 160Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His Pro Gln
Gly Ser Leu Asp Met Gly Asp Glu Ala 180 185
190Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu
Ser Glu 195 200 205Lys Val Val Asp
Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210
215 220Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser
Ser Leu Asp Val225 230 235
240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu
245 250 255Leu Gly Lys Lys Lys
Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260
265 270Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu
Gln Ser His Arg 275 280 285Pro Phe
Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 290
295 300Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys
Val Asn Ile Cys Cys305 310 315
320Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp
325 330 335Ile Ile Ala Pro
Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 340
345 350Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser
Leu Ser Phe His Ser 355 360 365Thr
Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 370
375 380Leu Lys Ser Cys Cys Val Pro Thr Lys Leu
Arg Pro Met Ser Met Leu385 390 395
400Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn
Met 405 410 415Ile Val Glu
Glu Cys Gly Cys Ser 4204305PRTHomo sapiens 4Met Pro Leu Leu
Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser
Glu Gly His Ser Ala Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro
35 40 45Asn Ser Gln Pro Glu Met Val Glu
Ala Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn
Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile
Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu
115 120 125Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu
Lys145 150 155 160Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His Pro Gln
Gly Ser Leu Asp Thr Gly Glu Glu Ala 180 185
190Glu Glu Val Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu
Ser Glu 195 200 205Lys Val Val Asp
Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 210
215 220Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser
Ser Leu Asp Val225 230 235
240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu
245 250 255Leu Gly Lys Lys Lys
Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 260
265 270Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu
Lys Glu Gln Ser 275 280 285His Arg
Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro 290
295 300His3055303PRTMus musculus 5Met Pro Leu Leu
Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser
Glu Gly His Gly Ser Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro
35 40 45Asn Ser Gln Pro Glu Met Val Glu
Ala Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn
Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile
Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu
115 120 125Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu
Lys145 150 155 160Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His Pro Gln
Gly Ser Leu Asp Thr Gly Asp Glu Ala 180 185
190Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu
Ser Glu 195 200 205Lys Val Val Asp
Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210
215 220Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser
Ser Leu Asp Val225 230 235
240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu
245 250 255Leu Gly Lys Lys Lys
Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260
265 270Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu
Gln Ser His Arg 275 280 285Pro Phe
Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 290
295 3006303PRTRattus norvegicus 6Met Pro Leu Leu Trp Leu
Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly
His Gly Ala Ala 20 25 30Pro
Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala
Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn
Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile
Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu
115 120 125Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu
Lys145 150 155 160Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His Pro Gln
Gly Ser Leu Asp Met Gly Asp Glu Ala 180 185
190Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu
Ser Glu 195 200 205Lys Val Val Asp
Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210
215 220Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser
Ser Leu Asp Val225 230 235
240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu
245 250 255Leu Gly Lys Lys Lys
Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260
265 270Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu
Gln Ser His Arg 275 280 285Pro Phe
Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 290
295 30075PRTArtificialSynthetic peptide 7Arg Arg Arg Arg
Arg1 58116PRTArtificialSynthetic peptide 8Gly Leu Glu Cys
Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe1 5
10 15Phe Val Ser Phe Lys Asp Ile Gly Trp Asn
Asp Trp Ile Ile Ala Pro 20 25
30Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile
35 40 45Ala Gly Thr Ser Gly Ser Ser Leu
Ser Phe His Ser Thr Val Ile Asn 50 55
60His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys65
70 75 80Cys Val Pro Thr Lys
Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp 85
90 95Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn
Met Ile Val Glu Glu 100 105
110Cys Gly Cys Ser 1159287PRTArtificialSynthetic peptide 9Met Pro
Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro
Gly Ser Glu Gly His Ser Ala Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val
Pro 35 40 45Asn Ser Gln Pro Glu
Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val
Pro Lys65 70 75 80Ala
Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly
85 90 95Glu Asn Gly Tyr Val Glu Ile
Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe
Ala Glu 115 120 125Ser Gly Thr Ala
Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130
135 140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp
Leu Phe Leu Lys145 150 155
160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His
Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180
185 190Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro
Val Ser Ser Ser 195 200 205Ile Gln
Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210
215 220Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser
Leu Val Leu Leu Gly225 230 235
240Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gly Gly
245 250 255Gly Glu Gly Gly
Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His Arg 260
265 270Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu
Asp His Pro His 275 280
28510285PRTArtificialSynthetic peptide 10Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly
Ser Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu
Ser Glu Lys Val 180 185 190Val
Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser Ser 195
200 205Ile Gln Arg Leu Leu Asp Gln Gly Lys
Ser Ser Leu Asp Val Arg Ile 210 215
220Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly225
230 235 240Lys Lys Lys Lys
Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp Gly 245
250 255Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu
Gln Ser His Arg Pro Phe 260 265
270Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275
280 28511285PRTArtificialSynthetic peptide 11Met
Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1
5 10 15Ile Val Arg Ser Ser Pro Thr
Pro Gly Ser Glu Gly His Gly Ala Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp
Gly Pro 35 40 45Asn Ser Gln Pro
Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro
Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly
85 90 95Glu Asn Gly Tyr Val Glu
Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile
Thr Phe Ala Glu 115 120 125Ser Gly
Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130
135 140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val
Trp Leu Phe Leu Lys145 150 155
160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys
His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180
185 190Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe
Pro Val Ser Ser Ser 195 200 205Ile
Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210
215 220Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala
Ser Leu Val Leu Leu Gly225 230 235
240Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp
Gly 245 250 255Ser Asp Gly
Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro Phe 260
265 270Leu Met Leu Gln Ala Arg Gln Ser Glu Asp
His Pro His 275 280
28512288PRTArtificialSynthetic peptide 12Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu
Leu Ser Glu Lys 180 185 190Val
Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser 195
200 205Ser Ile Gln Arg Leu Leu Asp Gln Gly
Lys Ser Ser Leu Asp Val Arg 210 215
220Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu225
230 235 240Gly Lys Lys Lys
Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gly 245
250 255Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu
Glu Lys Glu Gln Ser His 260 265
270Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His
275 280 28513286PRTArtificialSynthetic
peptide 13Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp
Ile1 5 10 15Ile Val Arg
Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20
25 30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr
Leu Pro Lys Asp Gly Pro 35 40
45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60Met Leu His Leu Lys Lys Arg Pro Asp
Val Thr Gln Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys
Val Gly 85 90 95Glu Asn
Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser
Glu Ile Ile Thr Phe Ala Glu 115 120
125Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly
130 135 140Ser Asp Leu Ser Val Val Glu
Arg Ala Glu Val Trp Leu Phe Leu Lys145 150
155 160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr
Ile Arg Leu Phe 165 170
175Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys
180 185 190Val Val Asp Ala Arg Lys
Ser Thr Trp His Ile Phe Pro Val Ser Ser 195 200
205Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp
Val Arg 210 215 220Ile Ala Cys Glu Gln
Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu225 230
235 240Gly Lys Lys Lys Lys Lys Glu Val Asp Gly
Asp Gly Lys Lys Lys Asp 245 250
255Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro
260 265 270Phe Leu Met Leu Gln
Ala Arg Gln Ser Glu Asp His Pro His 275 280
28514286PRTArtificialSynthetic peptide 14Met Pro Leu Leu Trp Leu
Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly
His Gly Ala Ala 20 25 30Pro
Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala
Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn
Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile
Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu
115 120 125Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu
Lys145 150 155 160Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His Gly Glu
Arg Ser Glu Leu Leu Leu Ser Glu Lys 180 185
190Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val
Ser Ser 195 200 205Ser Ile Gln Arg
Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210
215 220Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser
Leu Val Leu Leu225 230 235
240Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp
245 250 255Gly Ser Asp Gly Gly
Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro 260
265 270Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His
Pro His 275 280
28515289PRTArtificialSynthetic peptide 15Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu
Leu Leu Ser Glu 180 185 190Lys
Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 195
200 205Ser Ser Ile Gln Arg Leu Leu Asp Gln
Gly Lys Ser Ser Leu Asp Val 210 215
220Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu225
230 235 240Leu Gly Lys Lys
Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 245
250 255Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp
Glu Glu Lys Glu Gln Ser 260 265
270His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro
275 280
285His16287PRTArtificialSynthetic peptide 16Met Pro Leu Leu Trp Leu Arg
Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His
Gly Ser Ala 20 25 30Pro Asp
Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val
Lys Lys His Ile Leu Asn 50 55 60Met
Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala
Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly
Arg Arg Ala Glu 100 105 110Met
Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His
Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala
Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Glu Arg Ser
Glu Leu Leu Leu Ser Glu 180 185
190Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser
195 200 205Ser Ser Ile Gln Arg Leu Leu
Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215
220Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val
Leu225 230 235 240Leu Gly
Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys
245 250 255Asp Gly Ser Asp Gly Gly Leu
Glu Glu Glu Lys Glu Gln Ser His Arg 260 265
270Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro
His 275 280
28517287PRTArtificialSynthetic peptide 17Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu
Leu Leu Ser Glu 180 185 190Lys
Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 195
200 205Ser Ser Ile Gln Arg Leu Leu Asp Gln
Gly Lys Ser Ser Leu Asp Val 210 215
220Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu225
230 235 240Leu Gly Lys Lys
Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 245
250 255Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu
Lys Glu Gln Ser His Arg 260 265
270Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His
275 280 28518290PRTArtificialSynthetic
peptide 18Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp
Ile1 5 10 15Ile Val Arg
Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20
25 30Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala
Leu Pro Lys Asp Val Pro 35 40
45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60Met Leu His Leu Lys Lys Arg Pro Asp
Val Thr Gln Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys
Val Gly 85 90 95Glu Asn
Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser
Glu Ile Ile Thr Phe Ala Glu 115 120
125Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly
130 135 140Ser Asp Leu Ser Val Val Glu
Arg Ala Glu Val Trp Leu Phe Leu Lys145 150
155 160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr
Ile Arg Leu Phe 165 170
175Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser
180 185 190Glu Lys Val Val Asp Ala
Arg Lys Ser Thr Trp His Val Phe Pro Val 195 200
205Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser
Leu Asp 210 215 220Val Arg Ile Ala Cys
Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val225 230
235 240Leu Leu Gly Lys Lys Lys Lys Lys Glu Glu
Glu Gly Glu Gly Lys Lys 245 250
255Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln
260 265 270Ser His Arg Pro Phe
Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His 275
280 285Pro His 29019288PRTArtificialSynthetic peptide
19Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1
5 10 15Ile Val Arg Ser Ser Pro
Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys
Asp Gly Pro 35 40 45Asn Ser Gln
Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln
Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly
85 90 95Glu Asn Gly Tyr Val Glu
Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile
Thr Phe Ala Glu 115 120 125Ser Gly
Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130
135 140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val
Trp Leu Phe Leu Lys145 150 155
160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys
His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser 180
185 190Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp
His Ile Phe Pro Val 195 200 205Ser
Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210
215 220Val Arg Ile Ala Cys Glu Gln Cys Gln Glu
Ser Gly Ala Ser Leu Val225 230 235
240Leu Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys
Lys 245 250 255Lys Asp Gly
Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His 260
265 270Arg Pro Phe Leu Met Leu Gln Ala Arg Gln
Ser Glu Asp His Pro His 275 280
28520288PRTArtificialSynthetic peptide 20Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu
Leu Leu Leu Ser 180 185 190Glu
Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val 195
200 205Ser Ser Ser Ile Gln Arg Leu Leu Asp
Gln Gly Lys Ser Ser Leu Asp 210 215
220Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val225
230 235 240Leu Leu Gly Lys
Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys 245
250 255Lys Asp Gly Ser Asp Gly Gly Leu Glu Glu
Glu Lys Glu Gln Ser His 260 265
270Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His
275 280 28521408PRTArtificialSynthetic
peptide 21Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp
Ile1 5 10 15Ile Val Arg
Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20
25 30Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala
Leu Pro Lys Asp Val Pro 35 40
45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60Met Leu His Leu Lys Lys Arg Pro Asp
Val Thr Gln Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys
Val Gly 85 90 95Glu Asn
Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser
Glu Ile Ile Thr Phe Ala Glu 115 120
125Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly
130 135 140Ser Asp Leu Ser Val Val Glu
Arg Ala Glu Val Trp Leu Phe Leu Lys145 150
155 160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr
Ile Arg Leu Phe 165 170
175Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val
180 185 190Val Asp Ala Arg Lys Ser
Thr Trp His Val Phe Pro Val Ser Ser Ser 195 200
205Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val
Arg Ile 210 215 220Ala Cys Glu Gln Cys
Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly225 230
235 240Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu
Gly Lys Lys Lys Gly Gly 245 250
255Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His Arg
260 265 270Pro Phe Leu Met Leu
Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 275
280 285Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val
Asn Ile Cys Cys 290 295 300Lys Lys Gln
Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp305
310 315 320Ile Ile Ala Pro Ser Gly Tyr
His Ala Asn Tyr Cys Glu Gly Glu Cys 325
330 335Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu
Ser Phe His Ser 340 345 350Thr
Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 355
360 365Leu Lys Ser Cys Cys Val Pro Thr Lys
Leu Arg Pro Met Ser Met Leu 370 375
380Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met385
390 395 400Ile Val Glu Glu
Cys Gly Cys Ser 40522406PRTArtificialSynthetic peptide
22Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1
5 10 15Ile Val Arg Ser Ser Pro
Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys
Asp Gly Pro 35 40 45Asn Ser Gln
Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln
Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly
85 90 95Glu Asn Gly Tyr Val Glu
Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile
Thr Phe Ala Glu 115 120 125Ser Gly
Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130
135 140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val
Trp Leu Phe Leu Lys145 150 155
160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys
His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180
185 190Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe
Pro Val Ser Ser Ser 195 200 205Ile
Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210
215 220Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala
Ser Leu Val Leu Leu Gly225 230 235
240Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp
Gly 245 250 255Ser Asp Gly
Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro Phe 260
265 270Leu Met Leu Gln Ala Arg Gln Ser Glu Asp
His Pro His Arg Arg Arg 275 280
285Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys 290
295 300Gln Phe Phe Val Ser Phe Lys Asp
Ile Gly Trp Asn Asp Trp Ile Ile305 310
315 320Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly
Glu Cys Pro Ser 325 330
335His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val
340 345 350Ile Asn His Tyr Arg Met
Arg Gly His Ser Pro Phe Ala Asn Leu Lys 355 360
365Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu
Tyr Tyr 370 375 380Asp Asp Gly Gln Asn
Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val385 390
395 400Glu Glu Cys Gly Cys Ser
40523406PRTArtificialSynthetic peptide 23Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu
Ser Glu Lys Val 180 185 190Val
Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser Ser 195
200 205Ile Gln Arg Leu Leu Asp Gln Gly Lys
Ser Ser Leu Asp Val Arg Ile 210 215
220Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly225
230 235 240Lys Lys Lys Lys
Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp Gly 245
250 255Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu
Gln Ser His Arg Pro Phe 260 265
270Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg Arg Arg
275 280 285Arg Arg Gly Leu Glu Cys Asp
Gly Lys Val Asn Ile Cys Cys Lys Lys 290 295
300Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile
Ile305 310 315 320Ala Pro
Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser
325 330 335His Ile Ala Gly Thr Ser Gly
Ser Ser Leu Ser Phe His Ser Thr Val 340 345
350Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn
Leu Lys 355 360 365Ser Cys Cys Val
Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr 370
375 380Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln
Asn Met Ile Val385 390 395
400Glu Glu Cys Gly Cys Ser 40524409PRTArtificialSynthetic
peptide 24Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp
Ile1 5 10 15Ile Val Arg
Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20
25 30Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala
Leu Pro Lys Asp Val Pro 35 40
45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60Met Leu His Leu Lys Lys Arg Pro Asp
Val Thr Gln Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys
Val Gly 85 90 95Glu Asn
Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser
Glu Ile Ile Thr Phe Ala Glu 115 120
125Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly
130 135 140Ser Asp Leu Ser Val Val Glu
Arg Ala Glu Val Trp Leu Phe Leu Lys145 150
155 160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr
Ile Arg Leu Phe 165 170
175Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys
180 185 190Val Val Asp Ala Arg Lys
Ser Thr Trp His Val Phe Pro Val Ser Ser 195 200
205Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp
Val Arg 210 215 220Ile Ala Cys Glu Gln
Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu225 230
235 240Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly
Glu Gly Lys Lys Lys Gly 245 250
255Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His
260 265 270Arg Pro Phe Leu Met
Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275
280 285Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys
Val Asn Ile Cys 290 295 300Cys Lys Lys
Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp305
310 315 320Trp Ile Ile Ala Pro Ser Gly
Tyr His Ala Asn Tyr Cys Glu Gly Glu 325
330 335Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser
Leu Ser Phe His 340 345 350Ser
Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala 355
360 365Asn Leu Lys Ser Cys Cys Val Pro Thr
Lys Leu Arg Pro Met Ser Met 370 375
380Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn385
390 395 400Met Ile Val Glu
Glu Cys Gly Cys Ser 40525407PRTArtificialSynthetic peptide
25Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1
5 10 15Ile Val Arg Ser Ser Pro
Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25
30Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys
Asp Gly Pro 35 40 45Asn Ser Gln
Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln
Pro Val Pro Lys65 70 75
80Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly
85 90 95Glu Asn Gly Tyr Val Glu
Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100
105 110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile
Thr Phe Ala Glu 115 120 125Ser Gly
Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130
135 140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val
Trp Leu Phe Leu Lys145 150 155
160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys
His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys 180
185 190Val Val Asp Ala Arg Lys Ser Thr Trp His Ile
Phe Pro Val Ser Ser 195 200 205Ser
Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210
215 220Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly
Ala Ser Leu Val Leu Leu225 230 235
240Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys
Asp 245 250 255Gly Ser Asp
Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro 260
265 270Phe Leu Met Leu Gln Ala Arg Gln Ser Glu
Asp His Pro His Arg Arg 275 280
285Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys 290
295 300Lys Gln Phe Phe Val Ser Phe Lys
Asp Ile Gly Trp Asn Asp Trp Ile305 310
315 320Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu
Gly Glu Cys Pro 325 330
335Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr
340 345 350Val Ile Asn His Tyr Arg
Met Arg Gly His Ser Pro Phe Ala Asn Leu 355 360
365Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met
Leu Tyr 370 375 380Tyr Asp Asp Gly Gln
Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile385 390
395 400Val Glu Glu Cys Gly Cys Ser
40526407PRTArtificialSynthetic peptide 26Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu
Leu Ser Glu Lys 180 185 190Val
Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser 195
200 205Ser Ile Gln Arg Leu Leu Asp Gln Gly
Lys Ser Ser Leu Asp Val Arg 210 215
220Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu225
230 235 240Gly Lys Lys Lys
Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp 245
250 255Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys
Glu Gln Ser His Arg Pro 260 265
270Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg Arg
275 280 285Arg Arg Arg Gly Leu Glu Cys
Asp Gly Lys Val Asn Ile Cys Cys Lys 290 295
300Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp
Ile305 310 315 320Ile Ala
Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro
325 330 335Ser His Ile Ala Gly Thr Ser
Gly Ser Ser Leu Ser Phe His Ser Thr 340 345
350Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala
Asn Leu 355 360 365Lys Ser Cys Cys
Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr 370
375 380Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile
Gln Asn Met Ile385 390 395
400Val Glu Glu Cys Gly Cys Ser
40527410PRTArtificialSynthetic peptide 27Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu
Leu Leu Ser Glu 180 185 190Lys
Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 195
200 205Ser Ser Ile Gln Arg Leu Leu Asp Gln
Gly Lys Ser Ser Leu Asp Val 210 215
220Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu225
230 235 240Leu Gly Lys Lys
Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 245
250 255Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp
Glu Glu Lys Glu Gln Ser 260 265
270His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro
275 280 285His Arg Arg Arg Arg Arg Gly
Leu Glu Cys Asp Gly Lys Val Asn Ile 290 295
300Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp
Asn305 310 315 320Asp Trp
Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly
325 330 335Glu Cys Pro Ser His Ile Ala
Gly Thr Ser Gly Ser Ser Leu Ser Phe 340 345
350His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser
Pro Phe 355 360 365Ala Asn Leu Lys
Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser 370
375 380Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys
Lys Asp Ile Gln385 390 395
400Asn Met Ile Val Glu Glu Cys Gly Cys Ser 405
41028408PRTArtificialSynthetic peptide 28Met Pro Leu Leu Trp Leu Arg
Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His
Gly Ser Ala 20 25 30Pro Asp
Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val
Lys Lys His Ile Leu Asn 50 55 60Met
Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala
Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly
Arg Arg Ala Glu 100 105 110Met
Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His
Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala
Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Glu Arg Ser
Glu Leu Leu Leu Ser Glu 180 185
190Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser
195 200 205Ser Ser Ile Gln Arg Leu Leu
Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215
220Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val
Leu225 230 235 240Leu Gly
Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys
245 250 255Asp Gly Ser Asp Gly Gly Leu
Glu Glu Glu Lys Glu Gln Ser His Arg 260 265
270Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro
His Arg 275 280 285Arg Arg Arg Arg
Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys 290
295 300Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly
Trp Asn Asp Trp305 310 315
320Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys
325 330 335Pro Ser His Ile Ala
Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 340
345 350Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser
Pro Phe Ala Asn 355 360 365Leu Lys
Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 370
375 380Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys
Asp Ile Gln Asn Met385 390 395
400Ile Val Glu Glu Cys Gly Cys Ser
40529408PRTArtificialSynthetic peptide 29Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu
Leu Leu Ser Glu 180 185 190Lys
Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 195
200 205Ser Ser Ile Gln Arg Leu Leu Asp Gln
Gly Lys Ser Ser Leu Asp Val 210 215
220Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu225
230 235 240Leu Gly Lys Lys
Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 245
250 255Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu
Lys Glu Gln Ser His Arg 260 265
270Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg
275 280 285Arg Arg Arg Arg Gly Leu Glu
Cys Asp Gly Lys Val Asn Ile Cys Cys 290 295
300Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp
Trp305 310 315 320Ile Ile
Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys
325 330 335Pro Ser His Ile Ala Gly Thr
Ser Gly Ser Ser Leu Ser Phe His Ser 340 345
350Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe
Ala Asn 355 360 365Leu Lys Ser Cys
Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 370
375 380Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp
Ile Gln Asn Met385 390 395
400Ile Val Glu Glu Cys Gly Cys Ser
40530411PRTArtificialSynthetic peptide 30Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu
Leu Leu Leu Ser 180 185 190Glu
Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val 195
200 205Ser Ser Ser Ile Gln Arg Leu Leu Asp
Gln Gly Lys Ser Ser Leu Asp 210 215
220Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val225
230 235 240Leu Leu Gly Lys
Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys 245
250 255Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala
Asp Glu Glu Lys Glu Gln 260 265
270Ser His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His
275 280 285Pro His Arg Arg Arg Arg Arg
Gly Leu Glu Cys Asp Gly Lys Val Asn 290 295
300Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly
Trp305 310 315 320Asn Asp
Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu
325 330 335Gly Glu Cys Pro Ser His Ile
Ala Gly Thr Ser Gly Ser Ser Leu Ser 340 345
350Phe His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His
Ser Pro 355 360 365Phe Ala Asn Leu
Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met 370
375 380Ser Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile385 390 395
400Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 405
41031409PRTArtificialSynthetic peptide 31Met Pro Leu Leu Trp Leu
Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile1 5
10 15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly
His Gly Ser Ala 20 25 30Pro
Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala
Val Lys Lys His Ile Leu Asn 50 55
60Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn
Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85
90 95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile
Gly Arg Arg Ala Glu 100 105
110Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu
115 120 125Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135
140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu
Lys145 150 155 160Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe
165 170 175Gln Gln Gln Lys His Gly Gly
Gly Glu Arg Ser Glu Leu Leu Leu Ser 180 185
190Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe
Pro Val 195 200 205Ser Ser Ser Ile
Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210
215 220Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly
Ala Ser Leu Val225 230 235
240Leu Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys
245 250 255Lys Asp Gly Ser Asp
Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His 260
265 270Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu
Asp His Pro His 275 280 285Arg Arg
Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys 290
295 300Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp
Ile Gly Trp Asn Asp305 310 315
320Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu
325 330 335Cys Pro Ser His
Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His 340
345 350Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly
His Ser Pro Phe Ala 355 360 365Asn
Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 370
375 380Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile Gln Asn385 390 395
400Met Ile Val Glu Glu Cys Gly Cys Ser
40532409PRTArtificialSynthetic peptide 32Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile1 5 10
15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly
Ala Ala 20 25 30Pro Asp Cys
Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35
40 45Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys
Lys His Ile Leu Asn 50 55 60Met Leu
His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys65
70 75 80Ala Ala Leu Leu Asn Ala Ile
Arg Lys Leu His Val Gly Lys Val Gly 85 90
95Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg
Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115
120 125Ser Gly Thr Ala Arg Lys Thr Leu His Phe
Glu Ile Ser Lys Glu Gly 130 135 140Ser
Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys145
150 155 160Val Pro Lys Ala Asn Arg
Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165
170 175Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu
Leu Leu Leu Ser 180 185 190Glu
Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val 195
200 205Ser Ser Ser Ile Gln Arg Leu Leu Asp
Gln Gly Lys Ser Ser Leu Asp 210 215
220Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val225
230 235 240Leu Leu Gly Lys
Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys 245
250 255Lys Asp Gly Ser Asp Gly Gly Leu Glu Glu
Glu Lys Glu Gln Ser His 260 265
270Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His
275 280 285Arg Arg Arg Arg Arg Gly Leu
Glu Cys Asp Gly Lys Val Asn Ile Cys 290 295
300Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn
Asp305 310 315 320Trp Ile
Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu
325 330 335Cys Pro Ser His Ile Ala Gly
Thr Ser Gly Ser Ser Leu Ser Phe His 340 345
350Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro
Phe Ala 355 360 365Asn Leu Lys Ser
Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 370
375 380Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys
Asp Ile Gln Asn385 390 395
400Met Ile Val Glu Glu Cys Gly Cys Ser
405331167DNAArtificialSynthetic DNA 33agtcctactc ctggtagcga aggacattct
gctgctccag attgtccttc ttgtgctctt 60gctgctctgc ctaaggatgt gcctaattct
cagcctgaga tggttgaggc tgtgaagaaa 120cacatcctga acatgctgca cctgaagaaa
aggcctgatg tgactcagcc tgttcctaag 180gctgctttgc tgaacgctat taggaagctg
cacgttggta aggtgggaga gaatggttac 240gttgagatcg aggatgatat cggtagaagg
gctgagatga acgagctgat ggaacagacc 300tctgagatca tcaccttcgc tgagtctgga
accgctagaa agactctgca cttcgagatc 360agcaaagagg gtagcgatct gtctgttgtt
gagagggctg aggtgtggct tttcttgaag 420gtgccaaagg ctaataggac caggaccaag
gtgaccatta ggcttttcca acagcagaag 480cacgagagat ctgagttgct gctgtctgag
aaggttgtgg atgctagaaa gtccacctgg 540cacgttttcc ctgtgtcctc ttcaattcag
aggctgctgg atcagggtaa gagcagcctt 600gatgttagga ttgcttgcga gcagtgccaa
gagtctggtg cttctcttgt gcttctgggt 660aagaagaaaa agaaagagga agagggagaa
ggtaagaaaa agggtggtgg tgaaggtggt 720gctggtgctg atgaagagaa agagcagtct
cacaggcctt tcttgatgct tcaggctagg 780cagtctgagg atcaccctca cagaaggaga
agaaggggtc ttgagtgtga tggaaaggtg 840aacatctgct gcaagaagca gttcttcgtt
agcttcaagg atatcggttg gaacgattgg 900atcattgctc caagcggtta ccacgctaat
tactgtgagg gagagtgccc ttctcacatt 960gctggtacta gcggaagctc tctgtctttc
catagcaccg tgatcaacca ctacaggatg 1020aggggacata gccctttcgc taacctgaag
tcttgctgcg tgccaactaa gctgaggcct 1080atgtctatgc tgtactacga tgatggtcag
aacatcatca aaaaggatat ccagaacatg 1140atcgtggaag agtgcggttg ctcttag
1167341170DNAArtificialSynthetic DNA
34agtcctactc ctggtagcga aggacattct gctgctccag attgtccttc ttgtgctctt
60gctgctctgc ctaaggatgt gcctaattct cagcctgaga tggttgaggc tgtgaagaaa
120cacatcctga acatgctgca cctgaagaaa aggcctgatg tgactcagcc tgttcctaag
180gctgctttgc tgaacgctat taggaagctg cacgttggta aggtgggaga gaatggttac
240gttgagatcg aggatgatat cggtagaagg gctgagatga acgagctgat ggaacagacc
300tctgagatca tcaccttcgc tgagtctgga accgctagaa agactctgca cttcgagatc
360agcaaagagg gtagcgatct gtctgttgtt gagagggctg aggtgtggct tttcttgaag
420gtgccaaagg ctaataggac caggaccaag gtgaccatta ggcttttcca acagcagaag
480cacggtgaga gatctgagtt gctgctgtct gagaaggttg tggatgctag aaagtccacc
540tggcacgttt tccctgtgtc ctcttcaatt cagaggctgc tggatcaggg taagagcagc
600cttgatgtta ggattgcttg cgagcagtgc caagagtctg gtgcttctct tgtgcttctg
660ggtaagaaga aaaagaaaga ggaagaggga gaaggtaaga aaaagggtgg tggtgaaggt
720ggtgctggtg ctgatgaaga gaaagagcag tctcacaggc ctttcttgat gcttcaggct
780aggcagtctg aggatcaccc tcacagaagg agaagaaggg gtcttgagtg tgatggaaag
840gtgaacatct gctgcaagaa gcagttcttc gttagcttca aggatatcgg ttggaacgat
900tggatcattg ctccaagcgg ttaccacgct aattactgtg agggagagtg cccttctcac
960attgctggta ctagcggaag ctctctgtct ttccatagca ccgtgatcaa ccactacagg
1020atgaggggac atagcccttt cgctaacctg aagtcttgct gcgtgccaac taagctgagg
1080cctatgtcta tgctgtacta cgatgatggt cagaacatca tcaaaaagga tatccagaac
1140atgatcgtgg aagagtgcgg ttgctcttag
1170351173DNAArtificialSynthetic DNA 35agtcctactc ctggtagcga aggacattct
gctgctccag attgtccttc ttgtgctctt 60gctgctctgc ctaaggatgt gcctaattct
cagcctgaga tggttgaggc tgtgaagaaa 120cacatcctga acatgctgca cctgaagaaa
aggcctgatg tgactcagcc tgttcctaag 180gctgctttgc tgaacgctat taggaagctg
cacgttggta aggtgggaga gaatggttac 240gttgagatcg aggatgatat cggtagaagg
gctgagatga acgagctgat ggaacagacc 300tctgagatca tcaccttcgc tgagtctgga
accgctagaa agactctgca cttcgagatc 360agcaaagagg gtagcgatct gtctgttgtt
gagagggctg aggtgtggct tttcttgaag 420gtgccaaagg ctaataggac caggaccaag
gtgaccatta ggcttttcca acagcagaag 480cacggtggtg agagatctga gttgctgctg
tctgagaagg ttgtggatgc tagaaagtcc 540acctggcacg ttttccctgt gtcctcttca
attcagaggc tgctggatca gggtaagagc 600agccttgatg ttaggattgc ttgcgagcag
tgccaagagt ctggtgcttc tcttgtgctt 660ctgggtaaga agaaaaagaa agaggaagag
ggagaaggta agaaaaaggg tggtggtgaa 720ggtggtgctg gtgctgatga agagaaagag
cagtctcaca ggcctttctt gatgcttcag 780gctaggcagt ctgaggatca ccctcacaga
aggagaagaa ggggtcttga gtgtgatgga 840aaggtgaaca tctgctgcaa gaagcagttc
ttcgttagct tcaaggatat cggttggaac 900gattggatca ttgctccaag cggttaccac
gctaattact gtgagggaga gtgcccttct 960cacattgctg gtactagcgg aagctctctg
tctttccata gcaccgtgat caaccactac 1020aggatgaggg gacatagccc tttcgctaac
ctgaagtctt gctgcgtgcc aactaagctg 1080aggcctatgt ctatgctgta ctacgatgat
ggtcagaaca tcatcaaaaa ggatatccag 1140aacatgatcg tggaagagtg cggttgctct
tag 1173361176DNAArtificialSynthetic DNA
36agtcctactc ctggtagcga aggacattct gctgctccag attgtccttc ttgtgctctt
60gctgctctgc ctaaggatgt gcctaattct cagcctgaga tggttgaggc tgtgaagaaa
120cacatcctga acatgctgca cctgaagaaa aggcctgatg tgactcagcc tgttcctaag
180gctgctttgc tgaacgctat taggaagctg cacgttggta aggtgggaga gaatggttac
240gttgagatcg aggatgatat cggtagaagg gctgagatga acgagctgat ggaacagacc
300tctgagatca tcaccttcgc tgagtctgga accgctagaa agactctgca cttcgagatc
360agcaaagagg gtagcgatct gtctgttgtt gagagggctg aggtgtggct tttcttgaag
420gtgccaaagg ctaataggac caggaccaag gtgaccatta ggcttttcca acagcagaag
480cacggtggtg gtgagagatc tgagttgctg ctgtctgaga aggttgtgga tgctagaaag
540tccacctggc acgttttccc tgtgtcctct tcaattcaga ggctgctgga tcagggtaag
600agcagccttg atgttaggat tgcttgcgag cagtgccaag agtctggtgc ttctcttgtg
660cttctgggta agaagaaaaa gaaagaggaa gagggagaag gtaagaaaaa gggtggtggt
720gaaggtggtg ctggtgctga tgaagagaaa gagcagtctc acaggccttt cttgatgctt
780caggctaggc agtctgagga tcaccctcac agaaggagaa gaaggggtct tgagtgtgat
840ggaaaggtga acatctgctg caagaagcag ttcttcgtta gcttcaagga tatcggttgg
900aacgattgga tcattgctcc aagcggttac cacgctaatt actgtgaggg agagtgccct
960tctcacattg ctggtactag cggaagctct ctgtctttcc atagcaccgt gatcaaccac
1020tacaggatga ggggacatag ccctttcgct aacctgaagt cttgctgcgt gccaactaag
1080ctgaggccta tgtctatgct gtactacgat gatggtcaga acatcatcaa aaaggatatc
1140cagaacatga tcgtggaaga gtgcggttgc tcttag
117637432PRTArtificialSynthetic peptide 37His His His His His His Met Pro
Leu Leu Trp Leu Arg Gly Phe Leu1 5 10
15Leu Ala Ser Cys Trp Ile Ile Val Arg Ser Ser Pro Thr Pro
Gly Ser 20 25 30Glu Gly His
Ser Ala Ala Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala 35
40 45Leu Pro Lys Asp Val Pro Asn Ser Gln Pro Glu
Met Val Glu Ala Val 50 55 60Lys Lys
His Ile Leu Asn Met Leu His Leu Lys Lys Arg Pro Asp Val65
70 75 80Thr Gln Pro Val Pro Lys Ala
Ala Leu Leu Asn Ala Ile Arg Lys Leu 85 90
95His Val Gly Lys Val Gly Glu Asn Gly Tyr Val Glu Ile
Glu Asp Asp 100 105 110Ile Gly
Arg Arg Ala Glu Met Asn Glu Leu Met Glu Gln Thr Ser Glu 115
120 125Ile Ile Thr Phe Ala Glu Ser Gly Thr Ala
Arg Lys Thr Leu His Phe 130 135 140Glu
Ile Ser Lys Glu Gly Ser Asp Leu Ser Val Val Glu Arg Ala Glu145
150 155 160Val Trp Leu Phe Leu Lys
Val Pro Lys Ala Asn Arg Thr Arg Thr Lys 165
170 175Val Thr Ile Arg Leu Phe Gln Gln Gln Lys His Pro
Gln Gly Ser Leu 180 185 190Asp
Thr Gly Glu Glu Ala Glu Glu Val Gly Leu Lys Gly Glu Arg Ser 195
200 205Glu Leu Leu Leu Ser Glu Lys Val Val
Asp Ala Arg Lys Ser Thr Trp 210 215
220His Val Phe Pro Val Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly225
230 235 240Lys Ser Ser Leu
Asp Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser 245
250 255Gly Ala Ser Leu Val Leu Leu Gly Lys Lys
Lys Lys Lys Glu Glu Glu 260 265
270Gly Glu Gly Lys Lys Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp
275 280 285Glu Glu Lys Glu Gln Ser His
Arg Pro Phe Leu Met Leu Gln Ala Arg 290 295
300Gln Ser Glu Asp His Pro His Arg Arg Arg Arg Arg Gly Leu Glu
Cys305 310 315 320Asp Gly
Lys Val Asn Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe
325 330 335Lys Asp Ile Gly Trp Asn Asp
Trp Ile Ile Ala Pro Ser Gly Tyr His 340 345
350Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile Ala Gly
Thr Ser 355 360 365Gly Ser Ser Leu
Ser Phe His Ser Thr Val Ile Asn His Tyr Arg Met 370
375 380Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys
Cys Val Pro Thr385 390 395
400Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile
405 410 415Ile Lys Lys Asp Ile
Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 420
425 4303832DNAArtificialSynthetic DNA 38ccaacagcag
aagcacgaga gatctgagtt gc
323932DNAArtificialSynthetic DNA 39gcaactcaga tctctcgtgc ttctgctgtt gg
32
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